Summary The molecular and cellular regulation of fractures that do not heal is still not completely understood. However, deficiencies in numbers and osteogenic functions of mesenchymal stem cells (MSCs) are clearly associated with non-healing fractures. Accordingly, clinical studies conducted on exogenous addition of MSCs to enhance bone repair in segmental defects, nonunion of tibia and tibial osteotomy, have demonstrated encouraging results. However, lack of complete understanding of factors governing bone forming ability of MSCs and subsequent dearth of strategies thereof, to enhance bone forming potential of MSCs, have prevented utilization of optimal therapeutic potential of MSCs. It is recently reported that activation of toll like receptor 4 (TLR4) expressed on MSCs surface, polarizes MSCs into a pro-inflammatory phenotype called MSC1. The natural ligand of TLR4 is lipopolysaccharide (LPS), which is released at the injury site to recruit various cell types and this released LPS can potentially polarize MSCs into MSC1 phenotype. It is reported that in comparison with un-activated MSCs, MSC1 produces significantly more amount of cytokines (IL-6, IL-8, GM-CSF, IL-12 (promotes Th1 differentiation), TNF-?) that can recruit and activate T cells and it is less osteogenic. Therefore, inhibition of TLR4 signaling in MSCs might enhance their osteogenic potential. Recent discoveries have also revealed an astonishing link between local and systemic activation of T cells and inhibition of MSCs-mediated bone regeneration. Mice deficient in T cells display faster fracture healing than wild type mice and higher proportion of effector memory T cells, in patients? peripheral blood as well as fracture hematoma, correlates with delayed fracture healing. Natural antibodies (nIgM) are present in the blood since birth and play an important role in controlling functions of the immune cells. Human kidney and heart transplants performed in the subset of patients having high levels of nIgM were shown to have significantly lower incidence of acute rejections, thus permitting better graft survival. nIgM bind CD3, CD4 receptors on T cells to inhibit their activation and bind CCR5, CXCR4 to inhibit recruitment of T cells. nIgM also inhibit TLR4 signaling. This investigation explores the novel therapeutic use of nIgM to enhance bone regeneration. Aim 1 will test the hypothesis that nIgM will inhibit MSC1 phenotype through inhibition of TLR4 signaling and improve bone forming ability of MSCs. Aim 2 will test the hypothesis that nIgM will significantly diminish T cells inhibition of osteogenic MSCs in vitro and in vivo. Successful completion of the project will provide nIgM based revolutionary and simple method to improve bone healing in difficult fractures using MSCs.